Evolution and function of plant NO-Synthases – ALGAE-NOS

Nitric Oxide (NO) is a ubiquitous signaling mediator involved in many biological processes throughout the tree of life. In mammals, NO is central in the regulation of the vascular tone, the neural communication … and is synthesized by a family of enzymes, the NO-Synthases (NOSs). Most of the structural and functional investigations have focused on three mammalian NOS (mNOSs) that have become the standard NOSs. Hundreds of new NOS-like sequences have been recently found in the genome of organisms as diverse as bacteria, fungi, insects, corals... Despite the great heterogeneity of their structure, and the physiological diversity of their host organisms, these proteins have been considered as genuine “mammalian” NOSs. This mammal bias has led to the false and misleading assumption that all these proteins were designed to produce NO and exert signaling function. NO is known to be a major signaling molecule in plants, involved in processes as diverse as plant immunity, growth, N2-fixing symbiosis, or abiotic stresses response. The analysis of the land plants genome has established the absence of such NOSs in these organims. However, our teams (with others) have identified and described the presence of NOSs in green algae and characterized the structure and function of the first plant NOS. Algae-NOS is the prolongation of an ongoing collaboration that has successfully initiated the first investigations on this new NOS family. Our first results have highlighted the major structural variability of plant NOS structure, along with the great physiological diversity of their host algae and call for an in-depth investigation that tackles the complex relationship between NOS molecular structure, catalytic mechanism, environmental niche and biological function. In order to characterize the structure, activity and role of plant NOSs, Algae-NOS will develop an original approach that simultaneously combines modelling studies, structural characterization, and in vitro/in vivo functional investigations:
In silico: we will achieve an extensive inventory, structural classification and phylogenetic analysis of plant NOS family. We will select model NOSs reflecting the structural, physiological and phylogenetic diversity of NOSs in algae. We will generate 3D models of these NOSs by homology modeling and achieve a comparative analysis of the structural motifs that play critical role in NOS function or structure.
In vitro: the structure of the catalytic site of model NOSs and the electronic and structural properties of major catalytic intermediates will be characterized by a combination of biophysical spectroscopies. We will investigate the ability of these NOSs to synthesize NO but also to produce other Reactive Nitrogen Species in various milieu conditions. The structure-activity relationship of algae NOSs will allow us predicting their most relevant biochemical activity for distinct physiological settings.
In vivo: in vivo activity and role of plant NOSs will be investigated for a series of model organisms by coupling cellular and molecular biology approaches. The dynamic of expression and enzymatic activities of algae NOSs and the molecular basic of NOS-dependent signaling will be analyzed in processes such as immunity and abiotic stress responses. These results, along with the identification of the protein partners/targets will provide new information regarding the regulation and functions of NOSs in algae.
Such an interdisciplinary and integrative Structure-Activity-Environment approach is needed to characterize the molecular functioning and the biological role of plant NOSs. It will delineate the structural and physiological parameters that determine NOS activity, and help predicting their biological role in various physiological conditions. Beyond the function and evolution of NOS in plants, Algae-NOS will also provide valuable information to understand the role of NO in plants and the evolution of redox signalling in the green lineage.